Process for producing soy sauce having reduced busho-shu

ABSTRACT

The present invention relates to a process for producing a soy sauce koji having a reduced Busho-shu which is one of the unpleasant smells of soy sauce, and a soy sauce koji having a reduced Busho-shu obtained by this production process. Specifically, the process for producing a soy sauce koji is characterized by using a combination of a koji mold having a high isobutyric acid productivity and another koji mold having a low isobutyric acid productivity. The present invention also relates to a process for producing a soy sauce having a reduced Busho-shu and a soy sauce obtained by this production process.

TECHNICAL FIELD

The present invention relates to a process for producing a soy saucekoji having a reduced Busho-shu which is one of the unpleasant smells ofsoy sauce, and a soy sauce koji having a reduced Busho-shu obtained bythis production process. The present invention also relates to a processfor producing a soy sauce having a reduced Busho-shu and a soy sauceobtained by this production process.

BACKGROUND ART

In the production of soy sauce, firstly steam-cooked soybeans (ordefatted soybeans) and roasted and milled wheat in approximate quartersare mixed to obtain a raw material of soy sauce koji, which is thenadded and mixed with a seed koji mold belonging to the genus Aspergillus(such as Aspergillus oryzae and Aspergillus sojae). The mixture iscultured for 3 to 4 days under a controlled temperature in a koji-makingroom to obtain a soy sauce koji. Next, this soy sauce koji and a saltwater are mixed to obtain a soy sauce moromi mash, which is then placedin a tank for incubation. This soy sauce moromi mash is occasionallystirred to effect fermentation and aging to obtain an aged moromi mash.This aged moromi mash is wrapped in a nylon filter cloth andpress-filtered by a squeezer to obtain a raw soy sauce (a filtrate ofaged mash). This raw soy sauce is subjected to pasteurization,clarification, and sediment-removal to obtain a soy sauce (HakkoHandobukku (Fermentation Handbook), edited by Shinrokuro TOCHIKURA etal., Kyoritsu Shuppan Co. Ltd, 2001, pp. 588-592).

Moreover, a soy sauce is produced by the action of many microorganismssuch as koji molds (Aspergillus oryzae and Aspergillus sojae), salttolerant lactic acid bacteria (Tetragenococcus halophilus), salttolerant main fermentation yeast (Zygosaccharoinyces rouxii), and salttolerant after-ripening yeast (Candida versatilis and Candidaetchellsii) (Hakko Handobukku (Fermentation Handbook), edited byShinrokuro TOCHIKURA et al., as mentioned above).

Incidentally, soy sauce aroma is very complex and delicately differentdepending on the formula of raw material, koji mold, management ofkoji-making, lactic acid bacterium and yeast related to fermentation,intensity of fermentation, and the like. It is also known that the tasteis differently felt depending on this aroma (Nobuo SAITO, Jokyo, Vol.89, No. 7, pp. 498-500 (1994)). As to this soy sauce aroma, theexistence of aroma components which are said to be related to thepalatability and the freshness, namely furanones such as 4-hydroxy-2(or5)-ethyl-5(or 2)-methyl-3(2H)-furanone (hereunder, referred to as HEMF),alcohols such as 4-ethylguaiacol (4EG), 4-ethylphenol (4EP), methionol,isobutyl alcohol, and ethanol, esters such as ethyl acetate and ethyllactate, phenols, and sulfur-containing compounds, and conversely theexistence of components for Busho-shu (natto-like odor compounds) whichare unfavorable for soy sauce aroma and greatly spoil the quality,namely isobutyric acid and isovaleric acid are known (Tadanobu NAKADAI,Shoyu no Kenkyu to Gijutsu (Research and Technology of Soy Sauce), Vol.31, No. 4, pp. 223-233 (2005)).

Meanwhile, since soy sauce is a seasoning based on the umami taste ofamino acid, it is important in the production of soy sauce to increasethe production amount of amino acid and the production amount ofglutamic acid by increasing the utilization rate of nitrogen. Therefore,it is very important in the production of soy sauce to breed, search,and use koji molds that are capable of yielding strong protease andglutaminase and capable of producing a highly digestible koji.Conventionally, by such reasons, it has been considered to produce a soysauce koji (hereunder, also referred to as “koji-making”) by using aspecific koji mold which secretes a strong protease for a soy saucekoji.

As mentioned above, a process for producing a soy sauce koji whichproduce large amounts of amino acid and glutamic acid so as to produce asoy sauce having an excellent aroma and umami taste, has been desired.

DISCLOSURE OF THE INVENTION

In order to produce excellent soy sauce koji, koji molds which produce astrong protease and provide a highly digestible koji have beendeveloped. However, the use of such koji molds involves a risk in whicha high concentration of a component for Busho-shu which is one of theunpleasant smells of soy sauce, namely isobutyric acid, accumulates inthe raw material of soy sauce koji after 20 to 30 hours from the startof koji-making. This component for Busho-shu is generally metabolized(degraded) during the koji-making and hardly remains in the incubatedkoji (de-koji). However, in some cases depending on the koji moldstrain, this component may not be sufficiently metabolized during thekoji-making and remains as it is in the incubated koji (de-koji),resulting in that the smell is subsequently transferred to soy sauceproducts and negatively affects the soy sauce quality, in particular thearoma thereof.

Therefore, it is an objective of the present invention to produce a soysauce koji having a reduced Busho-shu which is one of the unpleasantsmells of soy sauce, and a soy sauce koji having a reduced Busho-shuusing the koji.

The inventors of the present invention have conducted intensive studiesto solve the above problems. As a result, they have found that, in thekoji-making by the inoculation of a koji mold having a high isobutyricacid productivity which belongs to the genus Aspergillus and a koji moldhaving a low isobutyric acid productivity which belongs to the genusAspergillus into a same raw material of soy sauce koji, the koji moldhaving a low isobutyric acid productivity metabolizes isobutyric acidthat has been produced and accumulated by the koji mold having a highisobutyric acid productivity, so as to provide a soy sauce koji havingless Busho-shu. Also, they have found that the production of a soy sauceusing this soy sauce koji provides a soy sauce having less Busho-shu.These findings have led to the completion of the present invention.

That is to say, the present invention relates to a process for producinga soy sauce koji having a reduced Busho-shu, wherein koji-making isperformed by inoculating a seed koji mold belonging to the genusAspergillus which produces a high concentration of isobutyric acidduring the koji-making and a seed koji mold belonging to the genusAspergillus which does not produce, or produces a low concentration ofisobutyric acid during the koji-making, into a same raw material of soysauce koji.

The koji mold belonging to the genus Aspergillus which produces a highconcentration of isobutyric acid during koji-making is preferably a kojimold which produces isobutyric acid at a double amount or more after 24or 30 hours from the start of koji-making, as compared with the amountof isobutyric acid produced after 20 hours from the start of thekoji-making. For example, the koji mold belonging to the genusAspergillus which produces a high concentration of isobutyric acidduring koji-making is a koji mold which produces 40 ppm or more ofisobutyric acid after 24 or 30 hours from the start of the koji-making,when koji-making is performed under the following condition (A):

Koji-Making Condition (A):

26 g of defatted soybeans is sprayed with 135 W/W % water. 28 g of wheatis roasted and milled, and the resultant is mixed with the thus obtainedsoybeans. The mixture is then put into a 2 liter Fernbach flask,followed by sterilization under pressure and heating at 121° C. for 50minutes. The resultant product is cooled down to a room temperature, andinoculated with 100 mg of bran seed koji. The flask is cotton-pluggedand then is incubated in a thermostatic chamber at a room temperature of30° C. to start koji-making. After 16 hours, the Fernbach flask isshaken so as to remove the heat generated in the koji (mixing processcalled “Teire”), and then is transferred into a thermostatic chamber ata room temperature of 25° C. After 8 hours, the “Teire” is performedagain to remove the heat generated in the koji, and then is transferredinto a thermostatic chamber at a room temperature of 20° C. Thekoji-making is performed for 42 hours in total to obtain a koji.

There is no limitation in the above koji mold belonging to the genusAspergillus which produces a high concentration of isobutyric acidduring koji-making, although Aspergillus sojae, and preferablyAspergillus sojae strain ATCC 46250, can be used.

On the other hand, the koji mold belonging to the genus Aspergilluswhich does not produce, or produces a low concentration of isobutyricacid during koji-making is preferably a koji mold which producesisobutyric acid at a reduced amount, or no isobutyric acid at all, after24 or 30 hours from the start of koji-making, as compared with theamount of isobutyric acid produced after 20 hours from the start of thekoji-making. For example, the koji mold belonging to the genusAspergillus which does not produce, or produces a low concentration ofisobutyric acid during koji-making is a koji mold which produces 20 ppmor less of isobutyric acid after 24 or 30 hours from the start of thekoji-making, when a koji-making is performed under said condition (A).

There is no limitation in the above koji mold belonging to the genusAspergillus which does not produce, or produces a low concentration ofisobutyric acid during koji-making, although Aspergillus oryzae, andpreferably Aspergillus oryzae strain ATCC 22787, can be used.

Moreover, the present invention relates to a soy sauce koji produced byany one of abovementioned processes and having a reduced Busho-shu.

Furthermore, the present invention relates to a process for producing asoy sauce having a reduced Busho-shu, wherein a soy sauce koji producedby any one of abovementioned processes is mixed with a salt water toproduce a soy sauce moromi mash, followed by fermentation and aging.

The present invention also relates to a soy sauce produced by theabovementioned process and having a reduced Busho-shu.

According to the present invention, a soy sauce koji and a soy saucehaving reduced Busho-shu can be reliably produced. Moreover, a soy saucehaving an extremely high content of glutamic acid and an excellent aromacan be produced at a high nitrogen utilization rate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of isobutyric acid contents (ppm) in koji overtime in the course of koji-making using Aspergillus sojae strain ATCC46250, Aspergillus oryzae strain ATCC 22787, and a mixture of these twotypes of strains.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be described in detail.

The present invention relates to a process for producing a soy saucekoji and a soy sauce having reduced Busho-shu. For the soy sauce koji,soybeans are used as a protein raw material and wheat is used as astarch raw material. The production thereof is carried out by mixingsoaked and steam-cooked soybeans, roasted and milled wheat, and a seedkoji, followed by koji-making. The present invention is characterized byusing koji molds having different isobutyric acid productivities as aseed koji in the course of koji-making so that the isobutyric acidcontent in the soy sauce koji is reduced so as to reduce a Busho-shu inthe koji.

Specifically, a koji mold belonging to the genus Aspergillus whichproduces a high concentration of isobutyric acid during koji-making(hereunder, also referred to as “koji mold having a high isobutyric acidproductivity”) and a koji mold belonging to the genus Aspergillus whichdoes not produce, or produces a low concentration of isobutyric acid(hereunder, also referred to as “koji mold having a low isobutyric acidproductivity”) are used as a seed koji in the course of koji-making.Here, the term “koji mold having a high isobutyric acid productivity”refers to a koji mold which produces isobutyric acid at a highconcentration to a degree that a Busho-shu is detectable from a soysauce koji when produced using this koji mold. Specifically, it refersto a koji mold which produces isobutyric acid at a double amount ormore, and more preferably a triple amount or more, after 24 or 30 hoursfrom the start of koji-making, as compared with the amount of isobutyricacid produced after about 20 hours from the start of the koji-making.More specifically, it is a koji mold which produces 40 ppm or more ofisobutyric acid within about 24 to 42 hours from the start ofkoji-making, for example, after 24 or 30 hours therefrom.

Koji-Making Condition (A):

26 g of defatted soybeans is sprayed with 135 W/W % water. 28 g of wheatis roasted and milled, and the resultant is mixed with the thus obtainedsoybeans. The mixture is then put into a 2 liter Fernbach flask,followed by sterilization under pressure and heating at 121° C. for 50minutes. The resultant product is cooled down to a room temperature, andinoculated with 100 mg of bran seed koji. The flask is cotton-pluggedand then is incubated in a thermostatic chamber at a room temperature of30° C. to start koji-making. After 16 hours, the Fernbach flask isshaken so as to remove the heat generated in the koji (mixing processcalled “Teire”), and then is transferred into a thermostatic chamber ata room temperature of 25° C. After 8 hours, the “Teire” is performedagain shaken to remove the heat generated in the koji, and then istransferred into a thermostatic chamber at a room temperature of 20° C.The koji-making is performed for 42 hours in total to obtain a koji.

Process for Preparing Bran Seed Koji:

20 g of bran is sprayed with 80 W/W % water. 5 g of the above product isput into a 150 ml conical flask, followed by sterilization underpressure and heating at 121° C. for 50 minutes. The resultant product iscooled down to a room temperature, and inoculated with 2 to 3 scoops ofa platinum loop with a koji mold which has been previously pure-culturedand isolated. The flask is incubated in a thermostatic chamber at atemperature of 30° C. for 72 hours.

On the other hand, the term “koji mold having a low isobutyric acidproductivity” refers to a koji mold which does not produce, or producesisobutyric acid at a low concentration to a degree that a Busho-shu isundetectable from a soy sauce koji when produced using this koji mold.Specifically, it refers to a koji mold which produces isobutyric acid ata reduced amount after 24 or 30 hours from the start of koji-making, ascompared with the amount of isobutyric acid produced after about 20hours from the start of the koji-making, or a koji mold which does notproduce isobutyric acid at all in the course of koji-making. Morespecifically, it is a koji mold which produces 20 ppm or less ofisobutyric acid within about 24 to 42 hours from the start ofkoji-making, for example, after 24 or 30 hours therefrom.

Koji molds having the abovementioned properties can be readily obtainedby preparing koji molds belonging to the genus Aspergillus, followed bykoji-making according to a normal koji-making condition, preferably theabove koji-making condition (A), measuring the amount of isobutyric acidin the koji, and selecting a strain which produces a large or smallamount of isobutyric acid.

Koji molds belonging to the genus Aspergillus to be prepared are notspecifically limited, although Aspergillus oryzae or Aspergillus sojaeis preferred considering that these molds are used for the production ofsoy sauce koji.

The isobutyric acid content in koji or soy sauce can be measured byusing any publicly known method. For example, 25 g of soy sauce koji isadded with 40 ml of saturated salt water, which is well shaken and leftat a room temperature for 24 hours. Then, the resultant product isfiltered with a filter paper. Thus obtained filtrate (soy sauce kojiextract) is extracted with methyl acetate, and is subjected to theanalysis by gas chromatography (GC) method without a concentrationprocess. Moreover, soy sauce is directly extracted as it is with methylacetate, and is subjected to the analysis by gas chromatography (GC)method without a concentration process (see “Analysis of AromaComponents by Gas Chromatography” edited and written by THE JAPAN SOYSAUCE INSPECTION INSTITUTE, Association of Experimental Method for SoySauce, issued on Mar. 1, 1985, pp. 177 to 179).

As to the koji mold having a high isobutyric acid productivity, forexample, Aspergillus sojae strain, preferably Aspergillus sojae strainATCC 46250 which has been confirmed to produce 60 ppm or more ofisobutyric acid during koji-making when the koji-making is performedunder the abovementioned condition (A), can be used. This strain ATCC46250 is deposited to the American Type Culture Collection, and isreadily available. Moreover, this Aspergillus sojae strain ATCC 46250 iscapable of producing a highly digestible soy sauce koji having a highprotease activity and a high glutamic acid content, which is thusparticularly preferably used in the present invention.

Moreover, as to the koji mold having a low isobutyric acid productivity,for example, Aspergillus oryzae strain, preferably Aspergillus oryzaestrain ATCC 22787 which has been confirmed to produce 10 ppm or less ofisobutyric acid during koji-making when the koji-making is performedunder the abovementioned condition (A), can be used. This strain ATCC22787 is also deposited to the American Type Culture Collection, and isreadily available.

In the present invention, a plurality of types of koji molds can beemployed so long as a koji mold having a high isobutyric acidproductivity and a koji mold having a low isobutyric acid productivityare used. For example, one type of koji mold having a high isobutyricacid productivity and one type of koji mold having a low isobutyric acidproductivity may be employed. Two types or more of koji molds havinghigh isobutyric acid productivities and one type of koji mold having alow isobutyric acid productivity may also be employed. Alternatively,one type of koji mold having a high isobutyric acid productivity and twotypes or more of koji molds having low isobutyric acid productivitiesmay be employed.

The amounts of koji mold having a high isobutyric acid productivity andkoji mold having a low isobutyric acid productivity to be used forkoji-making are not specifically limited. For example, the koji moldhaving a high isobutyric acid productivity and the koji mold having alow isobutyric acid productivity are mixed at a ratio of 20 to 80:80 to20, preferably 40 to 60:60 to 40, and more preferably 45 to 55: 55 to45.

The above koji mold having a high isobutyric acid productivity and kojimold having a low isobutyric acid productivity are used in accordancewith a normal process for producing a soy sauce koji. In a briefdescription, the seed koji as mentioned above may be inoculated in anormal raw material of koji, for example a raw material of soy saucekoji which is obtained by mixing water-sprayed and steam-cooked soybeanraw material with roasted and milled wheat raw material, followed bykoji-making (culturing) at about 25° C. to 35° C. for an appropriateperiod of time. At this time, the koji mold having a high isobutyricacid productivity and the koji mold having a low isobutyric acidproductivity can be inoculated in a same raw material of soy sauce koji,either concurrently or after a fixed interval. Other koji-makingconditions, such as the amount of koji mold to be used, the temperature,humidity, and time for culture, the mode of culture (such as continuoustype or batch type), the aeration-ventilation condition, and the numberof times and duration of stirring (Teire) are not specifically limited,and an appropriate and publicly known condition in the art can beselected.

The soy sauce koji obtained as mentioned above has a reduced content ofisobutyric acid, which is thus a soy sauce koji having a reducedBusho-shu. According to the process of the present invention, the amountof isobutyric acid contained in a koji is reduced to about 50% or less,and preferably about 30% or less, as compared to a soy sauce kojiobtained by a conventional production process. Moreover, a soy saucekoji obtained by the present invention is highly digestible, high in theglutamic acid content, and capable of producing a soy sauce having ahigh ratio (Glu/TN) of glutamic acid to the total nitrogen (TN).

Further, a soy sauce koji obtained by the present invention can be usedfor producing a soy sauce having a reduced Busho-shu. A soy sauce can beproduced using a soy sauce koji produced as mentioned above, through amoromi mash fermentation process and a press-filtration processaccording to a usual method. Specifically, a soy sauce koji is incubatedwith an appropriate concentration of salt water at a normal ratio forincubation, followed by fermentation and aging for 3 to 6 months withappropriate stirring by a usual method, to obtain an aged moromi mash.Next, the aged moromi mash is wrapped in a nylon filter cloth andpress-filtered by a squeezer to obtain a raw soy sauce. This raw soysauce is subjected to pasteurization, clarification, andsediment-removal to obtain a soy sauce. There is no specific limitationin the types of lactic acid bacterium and yeast to be used for themoromi mash fermentation process, and any appropriate and publicly knownones in the art may be employed.

According to the present invention, for the production of a soy saucekoji, with use of a koji mold which produces a strong protease andprovides a highly digestible koji, it is possible to avoid a risk inwhich a high concentration of a component for Busho-shu which is one ofthe unpleasant smells of soy sauce, namely isobutyric acid, accumulatesin the koji during the koji-making, and to reliably obtain a soy saucehaving a reduced Busho-shu. Moreover, according to the presentinvention, such a Busho-shu, which has been so far considered asunavoidable, can be reliably kept from being generated and accumulatedduring the koji-making, and the amount of isobutyric acid contained inthe soy sauce is reduced to about 60% or less, for example, about 50%.Therefore, according to the present invention, a soy sauce having areduced Busho-shu can be obtained. The process of the present inventionenables to produce a soy sauce having a high glutamic acid content andan excellent aroma.

EXAMPLES

The present invention is hereafter described in greater detail withreference to the following examples, although the technical scope of thepresent invention is not limited thereto.

Example 1 Process for Producing Soy Sauce Koji with Use of Mixed SeedKoji and Measurement of Isobutyric Acid Content (1) Use of Mixed SeedKoji of Aspergillus Sojae and Aspergillus Oryzae

26 g of defatted soybeans is sprayed with 135 W/W % water. 28 g of wheatis roasted and milled, and the resultant is mixed with the thus obtainedsoybeans. The mixture is then put into a 2 liter Fernbach flask,followed by sterilization under pressure and heating at 121° C. for 50minutes. The resultant product was cooled down to a room temperature,and inoculated with 200 mg of mixed seed koji containing 100 mg of seedkoji of Aspergillus sojae strain ATCC 46250 and 100 mg of seed koji ofAspergillus oryzae strain ATCC 22787. The flask was cotton-plugged andthen was incubated in a thermostatic chamber at a room temperature of30° C. to start koji-making. After 16 hours, the Fernbach flask wasshaken so as to remove the heat generated in the koji (mixing processcalled “Teire”), and then was transferred into a thermostatic chamber ata room temperature of 25° C. After 8 hours, the “Teire” was performedagain to remove the heat generated in the koji, and then was transferredinto a thermostatic chamber at a room temperature of 20° C. Thekoji-making was performed for 42 hours in total to obtain a soy saucekoji of the mixed seed koji.

The seed koji used herein was prepared by the following manner. 20 g ofbran was sprayed with 80 W/W % water. 5 g of the above product was putinto a 150 ml conical flask, followed by sterilization under pressureand heating at 121° C. for 50 minutes. The resultant product was cooleddown to a room temperature, and inoculated with 2 to 3 scoops of aplatinum loop with a koji mold which had been previously pure-culturedand isolated. The flask was incubated in a thermostatic chamber at atemperature of 30° C. for 72 hours (hereunder, the same manner wasperformed in comparative example and control example).

(2) Comparative Example Use of Seed Koji of Aspergillus Sojae StrainATCC 46250

A soy sauce koji of comparative example was obtained in the exactly samemanner as that of the above process for producing soy sauce koji (1),except for that “100 mg of seed koji of Aspergillus sojae strain ATCC46250” was used instead of the “mixed seed koji containing 100 mg ofseed koji of Aspergillus sojae strain ATCC 46250 and 100 mg of seed kojiof Aspergillus oryzae strain ATCC 22787”.

(3) Control Example Use of Seed Koji of Aspergillus Oryzae Strain ATCC22787

A soy sauce koji of control example was obtained in the exactly samemanner as that of the above process for producing soy sauce koji (1),except for that “100 mg of seed koji of Aspergillus oryzae strain ATCC22787” was used instead of the “mixed seed koji containing 100 mg ofseed koji of Aspergillus sojae strain ATCC 46250 and 100 mg of seed kojiof Aspergillus oryzae strain ATCC 22787”.

(4) Measurement of Isobutyric Acid Content

Regarding the abovementioned production examples for producing threetypes of soy sauce koji (1) to (3), the ups and downs of isobutyric acidcontents in these koji were measured over time. The isobutyric acidcontent was measured by the following manner. 25 g of soy sauce koji wasadded with 40 ml of saturated salt water, which was well shaken and leftat a room temperature for 24 hours. Then, the resultant product wasfiltered with a filter paper. Thus obtained filtrate (soy sauce kojiextract) was extracted with methyl acetate, and was subjected to theanalysis by gas chromatography (GC) method without a concentrationprocess.

The results are shown in FIG. 1, wherein the X axis indicates the time(hours) elapsed from the start of koji-making, and the Y axis indicatesthe isobutyric acid content (ppm) in soy sauce koji.

The results in FIG. 1 show that Aspergillus sojae strain ATCC 46250(comparative example) produced 40 ppm or more of isobutyric acid in kojiafter 24 hours from the start of koji-making, and accumulated 130 ppmthereof at maximum. Moreover, it is also found that isobutyric acid wascontained at a high concentration of 60 ppm after 42 hours (FIG. 1, abroken line with rhomboids).

On the other hand, Aspergillus oryzae strain ATCC 22787 (controlexample) produced a low concentration of the component for Busho-shu(isobutyric acid) at 10 ppm after 24 hours from the start ofkoji-making, and showed approximately zero value after 42 hours, whichindicates the disappearance thereof (FIG. 1, a two-dot chain line withsquares).

Moreover, it is also found that the use of the mixed seed koji havingAspergillus sojae strain ATCC 46250 and Aspergillus oryzae strain ATCC22787 mixed at equal amounts was able to reduce the isobutyric acidcontent to about one-third as compared to the single use of Aspergillussojae strain ATCC 46250 (comparative example) (FIG. 1, a solid line withtriangles).

(5) Properties of Soy Sauce Koji

The moisture content, the pH, the amount of protease (U/g of koji),Glu/TN (ratio of glutamic acid to the total nitrogen content), and thedigestibility of soy sauce koji of the mixed seed koji lot, thecomparative example lot, and the control example lot that had beenobtained by the above (1) to (3).

The pH and the protease amount (U/g of koji) were measured in accordancewith the “Experimental Method for Soy Sauce” (THE JAPAN SOY SAUCEINSPECTION INSTITUTE).

The digestibility and Glu/TN were measured by the methods describedbelow.

(Method for Measuring Digestibility)

12 g of Koji was weighed, which was added with 62.5 ml of 12% salt waterand 7.5 ml of toluene. The mixture was incubated to effect autodigestionat 37° C. for 7 days. The resultant product was filtered with a filterpaper. The digestibility was calculated by dividing the nitrogenconcentration in the filtrate by the total nitrogen concentration (inthe filtrate and the residual material on the filter paper).

(Method for Measuring Glu/TN)

The Glu/TN was calculated by dividing the glutamic acid concentration inthe filtrate of the above koji digest liquid by the nitrogenconcentration.

The results of the measurements carried out as mentioned above are shownin Table. 1 below.

TABLE 1 Analytical values of koji Moisture Protease Digestibility LotCharacteristic content [%] pH [U/g of koji] Glu/TN [%] Comparative A.sojae 44.2 7.23 261 0.67 82.7 example lot ATCC 46250 Control A. oryzae44.8 6.95 139 0.55 78.7 example lot ATCC 22787 Mixed seed Mixed seed45.7 7.19 248 0.61 84.2 koji lot koji mold

From the results of FIG. 1 and Table 1, it is found that the soy saucekoji of the comparative example lot using A. sojae alone (after 24 hoursfrom the start of koji-making) has advantages of being very high in theprotease activity, high in the ratio (Glu/TN) of glutamic acid (Glu) tothe total nitrogen (TN), and high in the digestibility, while itconversely has a disadvantage of being very high in the concentration ofisobutyric acid which is a causative component for Busho-shu.

Meanwhile, it is found that the soy sauce koji of the control examplelot using A. oryzae alone (same as the above) is very low in theconcentration of isobutyric acid which is a causative component forBusho-shu, while it has disadvantages of being low in both the proteaseactivity and the ratio (Glu/TN) of glutamic acid (Glu) to the totalnitrogen (TN).

On the other hand, it is found that the soy sauce koji of the mixed seedkoji lot using a mixture of A. sojae and A. oryzae (same as the above)has characteristics of being high in all of the protease activity, theratio (Glu/TN) of glutamic acid (Glu) to the total nitrogen (TN), andthe digestibility, being low in the isobutyric acid content which is themost important factor, and thus having less Busho-shu, and that such akoji is therefore very useful for brewing a soy sauce.

Example 2 Process for Producing Soy Sauce (1) Production of Soy Saucewith Use of Soy Sauce Koji Obtained by Using Mixed Seed Koji

300 g of defatted soybeans is sprayed with 150 W/W % water. 300 g ofwheat is roasted and milled, and the resultant is mixed with the thusobtained soybeans. The mixture is then subjected to sterilization underpressure and heating at 121° C. for 50 minutes. The resultant productwas cooled down to a room temperature, and inoculated with 2 g of mixedseed koji containing 1 g of seed koji of Aspergillus sojae strain ATCC46250 and 1 g of seed koji of Aspergillus oryzae strain ATCC 22787. Theproduct was filled in a flat wooden koji cover having a length of 58 cm,a width of 30 cm, and a depth of 6 cm.

This cover was placed in a thermostatic chamber at a room temperature of30° C. to start koji-making. The production of the seed koji was carriedout in the same manner as that of the Example 1 (1).

After 16 hours, the product was agitated by hand (“Teire”) so as toremove the heat generated in the koji, and then was transferred into athermostatic chamber at a room temperature of 25° C. After 8 hours, the“Teire” was performed again to remove the heat generated in the koji,and then was transferred into a thermostatic chamber at a roomtemperature of 20° C. The koji-making was performed for 30 hours intotal to obtain a soy sauce koji of the mixed seed koji.

0.9 kg of thus obtained soy sauce koji was added with 1.4 liter of saltwater. The mixture was placed in a small tank for incubation targeting asalt concentration of about 16%, followed by controlling of the moromimash for 5 months in accordance with a normal process for producing asoy sauce koji, and thereby an aged soy sauce moromi mash was obtained.

In order to accelerate lactic acid fermentation and alcoholfermentation, a lactic acid bacterium (Tetragenococcus halophilus)isolated from the soy sauce moromi mash was added at 1×10⁵ c.f.u./g ofmoromi mash on 14th day of the incubation, and a yeast(Zygosaccahromyces rouxii) isolated from the soy sauce moromi mash wasadded at 1×10⁵ c.f.u./g of moromi mash on 35th day of the incubation.

This aged soy sauce moromi mash was press-filtered by a small squeezerto obtain a raw soy sauce. This soy sauce was pasteurized at 80° C. for30 minutes and cooled down to a room temperature. The resultant soysauce was left still for 2 days, and sediments were removed therefrom toobtain a clear soy sauce with less Busho-shu.

(2) Comparative Example Lot

A soy sauce of comparative example lot was obtained in the exactly samemanner as that of the above process for producing soy sauce in (1),except for that “1 g of seed koji of Aspergillus sojae strain ATCC46250” was used instead of “2 g of the mixed seed koji containing 1 g ofseed koji of Aspergillus sojae strain ATCC 46250 and 1 g of seed koji ofAspergillus oryzae strain ATCC 22787”.

(3) Control Example Lot

A soy sauce of control example lot was obtained in the exactly samemanner as that of the above process for producing soy sauce in (1),except for that “1 g of seed koji of Aspergillus oryzae strain ATCC22787” was used instead of “2 g seed koji containing 1 g of seed koji ofAspergillus sojae strain ATCC 46250 and 1 g of seed koji of Aspergillusoryzae strain ATCC 22787”.

(4) Component Analysis of Soy Sauce

Each soy sauce of the mixed seed koji lot, the comparative example lot,and the control example lot produced in the above (1) to (3) wassubjected to component analysis of raw soy sauce, followed by sensoryevaluation of flavor of pasteurized and aged soy sauce.

The component analysis of soy sauce, that is to say, the measurement ofNaCl, TN (total nitrogen), Glu (glutamic acid), Glu/TN, RS (directreducing sugar), Alc (alcohol), Lac (lactic acid), pH, and Col (colornumber) in a soy sauce was in accordance with the “Experimental Methodfor Soy Sauce” (THE JAPAN SOY SAUCE INSPECTION INSTITUTE). Greater colornumbers mean lighter colors. The digestibility was obtained by thefollowing manner. 10 g of moromi mash was filtered with a filter paper,and then calculation was performed by dividing the “nitrogenconcentration in the filtrate” by the “total nitrogen amount in thefiltrate and the residual material on the filter paper”. The isobutyricacid content was obtained by the following manner. The soy sauce wasdirectly extracted as it was with methyl acetate, and was subjected tothe analysis by gas chromatography (GC) method without a concentrationprocess (see “Analysis of Aroma Components by Gas Chromatography” editedand written by THE JAPAN SOY SAUCE INSPECTION INSTITUTE, Association ofExperimental Method for Soy Sauce, issued on Mar. 1, 1985, pp. 177 to179).

The discrimination test and the preference test were performed by themethod of three-point discrimination and preference test (referred to astriangle test) (see “Method of Sensory Evaluation” edited and written byTHE JAPAN SOY SAUCE INSPECTION INSTITUTE, Association of ExperimentalMethod for Soy Sauce, issued on Mar. 1, 1985, pp. 117 to 118). Thismethod enables to perform both discrimination test and preference testin a single examination. First, a set consisting of three samples wasprepared by selecting two samples from one type of sample, and onesample from another type of sample. In the discrimination test, onesample (different type from other two samples) was selected among threesamples, followed by the preference test where comparison was performedbetween one selected sample and other two samples in terms ofpreference.

Table 2 shows the analytical values of the raw soy sauce components.Table 3 shows the results of the sensory evaluation. Table 4 shows theresults of discrimination and preference tests.

TABLE 2 Analytical values of raw soy sauce components NaCl TN Glu RS AlcLac Digestibility Lot Characteristic [%] [%] [%] Glu/TN [%] [%] [%] pHCol [%] Comparative A. sojae 16.25 1.81 1.10 0.61 2.72 3.85 0.79 4.88 3181.4 example lot ATCC 46250 Control A. oryzae 16.85 1.91 1.00 0.53 3.433.01 1.29 4.71 20 79.2 example lot ATCC 22787 Mixed seed Mixed koji moldof 16.33 1.88 1.26 0.67 2.76 3.63 0.87 4.92 28 81.1 koji lot A. sojaeand A. oryzae

TABLE 3 Results of sensory evaluation Charac- Isobutyric Lot teristicacid (ppm) Evaluation Comparative ATCC46250 20.4 unpleasant smellexample lot (A. sojae) Control ATCC22787 10.8 Excellent example lot (A.oryzae) Mixed seed koji lot Mixed koji mold 11.1 Excellent

TABLE 4 Results of discrimination and preference Discrimination testPreference test Number of Number of preference Number of correct amongpanelists who Lot panelists answers Judgment gave correct answersComparative 14 11 * 0 example lot Control 11 example lot Mixed seed 17 5— 3 koji lot Control 2 example lot The mark * means a significance of1%.

As shown in Table 2, it is found that the comparative example lot, thecontrol example lot, and the mixed seed koji lot provided almostequivalently excellent soy sauce in terms of the quality.

Moreover, as shown in Table 3, it is found that, in the results of thesensory evaluation, Busho-shu was felt in the comparative example lotfrom which about 20 ppm of isobutyric acid concentration had beendetected, while the aroma was excellent in both the control example lotand the mixed seed koji lot from which about 10 ppm of isobutyric acidconcentration had been detected.

Further, as shown in Table 4, it is found that, in the discriminationtest, the comparative example lot and the control example lot werediscriminated at a significance of 1%, and the comparative example lotwas prone to be disliked. On the other hand, it is found that the mixedseed koji lot and the control example lot were not discriminated.

From the above result, it is understood that, according to the presentinvention, even with use of a koji mold having a high isobutyric acidproductivity, a soy sauce having less unpleasant smell can be obtainedby koji-making in which another koji mold having a low isobutyric acidproductivity is additionally mixed therewith.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, a soy sauce koji and a soy saucehaving reduced Busho-shu can be reliably produced. Moreover, a soy saucehaving a high digestibility of koji, and a high content of glutamic acidcan be produced, and thus a soy sauce having excellent soy saucecomponents and an excellent aroma can be produced.

1. A process for producing a soy sauce koji having a reduced Busho-shu,wherein koji-making is performed by inoculating a seed koji moldbelonging to the genus Aspergillus which produces a high concentrationof isobutyric acid during the koji-making and a seed koji mold belongingto the genus Aspergillus which does not produce, or produces a lowconcentration of isobutyric acid during the koji-making, into a same rawmaterial of soy sauce koji.
 2. The process according to claim 1, whereinthe koji mold belonging to the genus Aspergillus which produces a highconcentration of isobutyric acid during koji-making is a koji mold whichproduces isobutyric acid at a double amount or more after 24 or 30 hoursfrom the start of koji-making, as compared with the amount of isobutyricacid produced after 20 hours from the start of the koji-making.
 3. Theprocess according to claim 1, wherein the koji mold belonging to thegenus Aspergillus which produces a high concentration of isobutyric acidduring koji-making is a koji mold which produces 40 ppm or more ofisobutyric acid after 24 or 30 hours from the start of the koji-making,when koji-making is performed under the following condition (A):Koji-making condition (A): 26 g of defatted soybeans is sprayed with 135W/W % water. 28 g of wheat is roasted and milled, and the resultant ismixed with the thus obtained soybeans. The mixture is then put into a 2liter Fernbach flask, followed by sterilization under pressure andheating at 121° C. for 50 minutes; the resultant product is cooled downto a room temperature, and inoculated with 100 mg of bran seed koji; theflask is cotton-plugged and then is incubated in a thermostatic chamberat a room temperature of 30° C. to start koji-making; after 16 hours,the Fernbach flask is shaken so as to remove the heat generated in thekoji (mixing process called “Teire”), and then is transferred into athermostatic chamber at a room temperature of 25° C.; after 8 hours, the“Teire” is performed again to remove the heat generated in the koji, andthen is transferred into a thermostatic chamber at a room temperature of20° C.; and the koji-making is performed for 42 hours in total to obtaina koji.
 4. The process according to claim 1, wherein the koji moldbelonging to the genus Aspergillus which produces a high concentrationof isobutyric acid during koji-making is Aspergillus sojae.
 5. Theprocess according to claim 1, wherein the koji mold belonging to thegenus Aspergillus which produces a high concentration of isobutyric acidduring koji-making is Aspergillus sojae strain ATCC
 46250. 6. Theprocess according to claim 1, wherein the koji mold belonging to thegenus Aspergillus which does not produce, or produces a lowconcentration of isobutyric acid during koji-making is a koji mold whichproduces isobutyric acid at a reduced amount, or no isobutyric acid atall, after 24 or 30 hours from the start of koji-making, as comparedwith the amount of isobutyric acid produced after 20 hours from thestart of the koji-making.
 7. The process according to claim 1, whereinthe koji mold belonging to the genus Aspergillus which does not produce,or produces a low concentration of isobutyric acid during koji-making isa koji mold which produces 20 ppm or less of isobutyric acid after 24 or30 hours from the start of the koji-making, when a koji-making isperformed under said condition (A).
 8. The process according to claim 1,wherein the koji mold belonging to the genus Aspergillus which does notproduce, or produces a low concentration of isobutyric acid duringkoji-making is Aspergillus oryzae.
 9. The process according to claim 8,wherein the koji mold belonging to the genus Aspergillus which does notproduce, or produces a low concentration of isobutyric acid duringkoji-making is Aspergillus oryzae strain ATCC
 22787. 10. A soy saucekoji produced by the process according to claim 1 and having a reducedBusho-shu.
 11. A process for producing a soy sauce having a reducedBusho-shu, wherein a soy sauce koji produced by the process according toclaim 1 is mixed with a salt water to produce a soy sauce moromi mash,followed by fermentation and aging.
 12. A soy sauce produced by theprocess according to claim 11 and having a reduced Busho-shu.